The Plasmodium falciparum eIK1 kinase (PfeIK1) is central for melatonin synchronization in the human malaria parasite. Melatotosil blocks melatonin action on parasite cell cycle.

Melatonin and its indoles derivatives are central in the synchronization of malaria parasites. In this research, we discovered that melatonin is unable to increase the parasitemia in the human malaria Plasmodium falciparum that lacks the kinase PfeIK1. The PfeIK1 knockout strain is a valuable tool in the screening of indol-related compound that blocks the melatonin effect in wild-type (WT) parasite development. The assays were performed by using flow cytometry with simultaneous labeling for mitochondria viability with MitoTracker Deep Red and nucleus staining with SYBR Green. We found that Melatotosil leads to an increase in parasitemia in P. falciparum and blocks melatonin effect in the WT parasite. Using microscopy imaging system, we found that Melatotosil at 500 nM is able to induce cytosolic calcium rise in transgenic PfGCaMP3 parasites. On the contrary, the compound Triptiofen blocks P. falciparum cell cycle with IC50 9.76 µM ± 0.6, inhibits melatonin action, and does not lead to a cytosolic calcium rise in PfGCaMP3 parasites. We also found that the synthetic indol-related compounds arrested parasite cycle for PfeIK1 knockout and (WT) P. falciparum (3D7) in 72 hours culture assays with the IC50 values slighting lower for the WT strain. We concluded that the kinase PfeIK1 is central for melatonin downstream signaling pathways involved in parasite cell cycle progression. More importantly, the indol-related compounds block its cycle as an upstream essential mechanism for parasite survival. Our data clearly show that this class of compounds emerge as an alternative for the problem of resistance with the classical antimalarials.

CCL19 suppresses angiogenesis through promoting miR-206 and inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway in colorectal cancer.

The mechanisms underlying the role of chemokines in tumor angiogenesis is still not fully understood. In this study, we detected the influence of CCL19 on colorectal cancer (CRC) angiogenesis. The expression of CCL19 and CD31 in CRC tissues were detected by immunohistochemistry. Human CRC cell lines SW1116 and SW620 stably transfected with CCL19 lentivirus and CCL19 shRNA, and HUVEC stably transfected with CCR7 shRNA were used in our study. Our study showed that CCL19 was significantly low-expressed in CRC tissues and positively related to highly tumor microvessel density. In vitro, we observed that CCL19 high-expressed SW1116 supernatant was able to inhibit proliferation, migration, and sprouting responses of HUVEC, whereas CCL19 low-expressed SW620 supernatant can promote HUVEC angiogenesis. Additionally, we further demonstrated that these functions maybe achieved through promoting miR-206 thus inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway in a CCR7-dependent manner. Mice angiogenesis model also confirmed that elevated expression of CCL19 inhibit the angiogenesis of CRC in vivo. In summary, our results supported that CCL19 can inhibit CRC angiogenesis through promoting miR-206 thus inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway. This may be a novel therapeutic option for anti-vascular treatment in CRC.

ETS-domain containing protein (Elk1) suppression protects cortical neurons against oxygen-glucose deprivation injury.

ETS-domain containing protein (Elk1), which is a transcription factor, is reported to be closely related to the apoptosis of primary neurons and could be activated by hypoxia in human microvascular endothelial cells. In this study, we aimed to investigate the role of Elk1 in cortical neurons under oxygen-glucose deprivation (OGD) conditions. The OGD model of cortical neurons was established the anoxia/hypoglycemia-induced injury and the in vivo model was established by middle cerebral artery occlusion (MCAO). Elk1 mRNA and protein expression was significantly up-regulated in neurons exposed to OGD for 24 h, and mRNA expression was also markedly increased in cerebral cortex of rats with MCAO after 10 days. The knockdown of Elk1 in neurons without OGD obviously constrained Fra-1 and promoted Nrf2 expression. Also, Elk1 inhibition suppressed neuronal apoptosis, caspase-3 activity, LDH leakage, and MDA and SOD contents, while it increased cell viability in the neurons with OGD. The overexpression of Fra-1 showed a reverse effect on caspase-3 activity, cell viability and SOD contents in neurons under OGD conditions compared with Elk1 knockdown. Thus, Elk1 inhibition has a protective effect on neurons against OGD-induced injury.

Disinhibition of the extracellular-signal-regulated kinase restores the amplification of circadian rhythms by lithium in cells from bipolar disorder patients.

UNLABELLED: Bipolar disorder (BD) is characterized by depression, mania, and circadian rhythm abnormalities. Lithium, a treatment for BD stabilizes mood and increases circadian rhythm amplitude. However, in fibroblasts grown from BD patients, lithium has weak effects on rhythm amplitude compared to healthy controls. To understand the mechanism by which lithium differentially affects rhythm amplitude in BD cells, we investigated the extracellular-signal-regulated kinase (ERK) and related signaling molecules linked to BD and circadian rhythms. In fibroblasts from BD patients, controls and mice, we assessed the contribution of the ERK pathway to lithium-induced circadian rhythm amplification. Protein analyses revealed low phospho-ERK1/2 (p-ERK) content in fibroblasts from BD patients vs. CONTROLS: Pharmacological inhibition of ERK1/2 by PD98059 attenuated the rhythm amplification effect of lithium, while inhibition of two related kinases, c-Jun N-terminal kinase (JNK), and P38 did not. Knockdown of the transcription factors CREB and EGR-1, downstream effectors of ERK1/2, reduced baseline rhythm amplitude, but did not alter rhythm amplification by lithium. In contrast, ELK-1 knockdown amplified rhythms, an effect that was not increased further by the addition of lithium, suggesting this transcription factor may regulate the effect of lithium on amplitude. Augmentation of ERK1/2 signaling through DUSP6 knockdown sensitized NIH3T3 cells to rhythm amplification by lithium. In BD fibroblasts, DUSP6 knockdown reversed the BD rhythm phenotype, restoring the ability of lithium to increase amplitude in these cells. We conclude that the inability of lithium to regulate circadian rhythms in BD may reflect reduced ERK activity, and signaling through ELK-1.

Type II cGMP-dependent protein kinase inhibits ERK/JNK-mediated activation of transcription factors in gastric cancer cells.

A previous study has shown that type II cGMP­dependent protein kinase (PKG II) inhibits the proliferation of gastric cancer cells through blocking EGF-triggered MAPK/ERK signal transduction, indicating that the kinase may be a potential anticancer factor. In the present study, the role of PKG II in the EGF-induced activation of transcription factors in the MAPK/ERK signal transduction pathway was investigated. BGC-823 human gastric cancer cells were infected with adenoviral constructs encoding the cDNA of PKG II (pAd­PKG II) to increase the expression of PKG II and treated with 8-pCPT­cGMP to activate the enzyme. Using luciferase reporter assays, it was revealed that PKG II markedly suppressed the EGF-induced transcriptional activities of AP-1 and Elk1. Consistent with the inhibitory effect of PKG II on AP-1 activity, the expression levels of c-Jun and c-Fos, components of AP-1, were also inhibited. Co-immunoprecipitation analysis demonstrated that EGF treatment increased the AP-1 content through inducing the formation of p-c-Jun-c-Jun homodimers and p-c-Jun-c-Fos heterodimers. However, this combination was efficiently blocked by activated PKG II. While pretreatments with MAPK inhibitors suppressed the EGF-induced transcriptional activities of AP-1 and Elk1, PKG II prevented the EGF-induced phosphorylation/activation of ERK and JNK, but not the phosphorylation of p38MAPK induced by EGF. These data suggest that PKG II inhibits the EGF-triggered proliferation of gastric cancer cells through suppressing ERK-/JNK-, but not p38MAPK, -mediated AP-1 and Elk1 transactivation.

Alterations of molecular and behavioral responses to cocaine by selective inhibition of Elk-1 phosphorylation.

Activation of the extracellular signal-regulated kinase (ERK) signaling pathway in the striatum is crucial for molecular adaptations and long-term behavioral alterations induced by cocaine. In response to cocaine, ERK controls the phosphorylation levels of both mitogen and stress-activated protein kinase 1 (MSK-1), a nuclear kinase involved in histone H3 (Ser10) and cAMP response element binding protein phosphorylation, and Elk-1, a transcription factor involved in serum response element (SRE)-driven gene regulations. We recently characterized the phenotype of msk-1 knock-out mice in response to cocaine. Herein, we wanted to address the role of Elk-1 phosphorylation in cocaine-induced molecular, morphological, and behavioral responses. We used a cell-penetrating peptide, named TAT-DEF-Elk-1 (TDE), which corresponds to the DEF docking domain of Elk-1 toward ERK and inhibits Elk-1 phosphorylation induced by ERKs without modifying ERK or MSK-1 in vitro. The peptide was injected in vivo before cocaine administration in mice. Immunocytochemical, molecular, morphological, and behavioral studies were performed. The TDE inhibited Elk-1 and H3 (Ser10) phosphorylation induced by cocaine, sparing ERK and MSK-1 activation. Consequently, TDE altered cocaine-induced regulation of genes bearing SRE site(s) in their promoters, including c-fos, zif268, ΔFosB, and arc/arg3.1 (activity-regulated cytoskeleton-associated protein). In a chronic cocaine administration paradigm, TDE reversed cocaine-induced increase in dendritic spine density. Finally, the TDE delayed the establishment of cocaine-induced psychomotor sensitization and conditioned-place preference. We conclude that Elk-1 phosphorylation downstream from ERK is a key molecular event involved in long-term neuronal and behavioral adaptations to cocaine.

MEK-ERK pathway regulates EZH2 overexpression in association with aggressive breast cancer subtypes.

EZH2 overexpression occurs in various malignancies and is associated with a poor outcome. We have so far demonstrated that EZH2 downregulates the important genes such as E-cadherin and RUNX3 by increasing histone H3K27 trimethylation. However, the mechanism of EZH2 overexpression in various cancer cells remains unclear. In this study we carried out a promoter analysis of the EZH2 gene and investigated whether a survival signal that is upregulated in cancer cells is related to overexpression at the transcription level. We also explored the clinical relevance of the signaling pathway that leads to EZH2 overexpression in breast cancer and demonstrated that MEK-ERK1/2-Elk-1 pathway leads to EZH2 overexpression. The triple-negative and ERBB2-overexpressing subtypes of breast cancer are known to contain more rapidly proliferating breast cancer cells. The signaling pathway connected to EZH2 overexpression was associated with both aggressive subtypes of breast cancer. We show the significance that overexpression of histone modifier protein EZH2 in cancer cells and our study could pave the way for EZH2 inhibition to become an efficient treatment for more aggressive breast cancers.

Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer.

Myeloid cell leukaemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. In breast tumours, increased Mcl-1 expression correlates with high tumour grade and poor patient survival. We have previously demonstrated that Her-2 levels correspond to increased Mcl-1 expression in breast tumours. Epidermal growth factor (EGF) receptor signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival. Herein, we determined the critical downstream signals responsible for the EGF mediated increase of Mcl-1 and their role in cell survival. We found that both Mcl-1 mRNA and protein levels are rapidly induced upon stimulation with EGF. Promoter analysis revealed that an Elk-1 transcription factor-binding site is critical for EGF activation of the Mcl-1 promoter. Furthermore, we found that knockdown of Elk-1 or inhibition of the Erk signalling pathway was sufficient to block EGF upregulation of Mcl-1 and EGF mediated cell survival. Using chromatin immunoprecipitation and biotin labelled probes of the Mcl-1 promoter, we found that Elk-1 and serum response factor are bound to the promoter after EGF stimulation. To determine whether Mcl-1 confers a survival advantage, we found that knockdown of Mcl-1 expression increased apoptosis whereas overexpression of Mcl-1 inhibited drug induced cell death. In human breast tumours, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels. These results indicate that the EGF induced activation of Elk-1 is an important mediator of Mcl-1 expression and cell survival and therefore a potential therapeutic target in breast cancer.

AC3-33, a novel secretory protein, inhibits Elk1 transcriptional activity via ERK pathway.

The transcription factor AP-1 plays an important role in cellular proliferation, transformation and death. In this study, we report a novel human gene, AC3-33 (GenBank name: c3orf33, FLJ31139), which encodes a secretory protein that can inhibit Elk1 transcriptional activity via ERK1/2 pathway. The AC3-33 mRNA encodes a protein of 251 amino acids, which is a classical secretory protein. Functional investigation reveals that overexpression of AC3-33 significantly inhibit AP-1 activity and DNA-binding ability. Further investigation indicated that overexpression of AC3-33 significantly inhibit transcriptional activity of Elk1 and c-jun, but not c-fos. As for the upstream of signaling pathway of Elk-1, our study demonstrated that overexpression of AC3-33 significantly down-regulates phosphorylation of ERK1/2, but not JNK/SAPK or p38 MAPK. These results clearly indicate that AC3-33 is a novel member of the secretory family and inhibits Elk1 transcriptional activity via ERK1/2 MAPK.

Mitogen-induced recruitment of ERK and MSK to SRE promoter complexes by ternary complex factor Elk-1.

Many eukaryotic genes are acutely regulated by extra-cellular signals. The c-fos serum response element (SRE) mediates transcriptional activation in response to mitogens through serum response factor (SRF)-dependent recruitment of Elk-1, a mitogen-activated protein kinase (MAPK)-responsive transcription factor. How subsequent events at SRE promoters stimulate initiation of transcription has yet to be fully resolved. Here we show that extra-cellular signal-regulated kinase (ERK) and mitogen and stress-activated kinase (MSK) are recruited to SRE promoter complexes in vitro and in vivo. Their recruitment in vitro correlates with Elk-1 binding and for ERK the D domain/KIM of Elk-1 is specifically involved. In vivo, recruitment of ERK and MSK is stimulated by mitogens, correlates with histone H3 phosphorylation and is impaired by Elk-1 knockdown. Immunocytochemistry and confocal microscopy reveal that ERK appears to associate to some extent with initiating rather than elongating RNA polymerase II. Taken together, our data add to the body of evidence implying that ERK and related MAPKs may fulfil a generic role at the promoters of acutely regulated genes.

4-Hydroxy-2-nonenal adduction of extracellular signal-regulated kinase (Erk) and the inhibition of hepatocyte Erk-Est-like protein-1-activating protein-1 signal transduction.

4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation (LPO) product formed during oxidative stress. 4-HNE is highly reactive toward cellular nucleophiles and is implicated in the evolution of numerous pathologies associated with oxidative stress and LPO. Recent evidence suggests that chronic prooxidant exposure results in the loss of extracellular signal-regulated kinase (Erk)-1/2 phosphorylation in vivo, a signaling pathway associated with cellular proliferation, survival, and homeostasis. Immunodetection and molecular analysis were used in this study to evaluate the hypothesis that 4-HNE modification of Erk-1/2 inhibits constitutive Erk-Est-like protein (Elk)-1-activating protein (AP)-1 signaling. Primary rat hepatocytes treated with subcytotoxic, pathologically relevant concentrations of 4-HNE demonstrated a concentration-dependent loss of constitutive Erk-1/2 phosphorylation, activity, and nuclear localization. These findings were consistent with iron-induced intracellular LPO, which also resulted in a concentration-dependent decrease in hepatocyte Erk-1/2 phosphorylation and activity. 4-HNE and iron-induced inhibition of Erk-1/2 was inversely correlated with the accumulation of 4-HNE-Erk-1/2 monomer adducts. 4-HNE treatment of hepatocytes decreased nuclear total and phosphorylated Erk-1/2, Elk-1, and AP-1 phosphorylation as well as cFos and cJun activities. The cytosolic modification of unphosphorylated Erk-1/2 was evaluated in vitro using molar ratios of inactive Erk-2 to 4-HNE consistent with increasing oxidative stress in vivo. Liquid chromatography combined with tandem mass spectrometry confirmed monomer adduct formation and identified the major adduct species at the histidine 178 residue within the kinase phosphorylation lip. These novel results show that the formation of 4-HNE-Erk-1/2 monomer-adducts results in the inhibition of Erk-Elk-AP-1 signaling in hepatocytes and implicates the His 178 residue with the mechanism of inhibition.

Role of ETS transcription factors in the hypoxia-inducible factor-2 target gene selection.

Tumor hypoxia often directly correlates with aggressive phenotype, metastasis progression, and resistance to chemotherapy. Two transcription factors [hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha] are dramatically induced in hypoxic areas and regulate the expression of genes necessary for tumor adaptation to the conditions of low oxygen; however, the relative contribution of these factors is controversial. We used RNA interference-mediated inactivation of HIF-1alpha or HIF-2alpha followed by microarray analysis to identify genes specifically regulated by either HIF-1 or HIF-2 in hypoxia. We found that, in the MCF7 cell line, the vast majority of hypoxia-responsive genes (>80%) were dependent on the presence of HIF-1alpha. However, a small group of genes were preferentially regulated by HIF-2alpha. Promoter analysis for this group of genes revealed that all of them have putative binding sites for ETS family transcription factors, and 10 of 11 HIF-2alpha-dependent genes had at least one potential hypoxia-responsive element (HRE) in proximity to an ETS transcription factor binding site. Knockdown of ELK-1, the most often represented member of ETS family, significantly reduced hypoxic induction of the HIF-2alpha-dependent genes. Physical and functional interaction between ELK-1 and HIF-2alpha were supported by coimmunoprecipitation of these two proteins, luciferase reporter assay using CITED2 promoter, and binding of ELK-1 protein to the promoters of CITED2 and WISP2 genes in proximity to a HRE. These data suggest that the choice of the target genes by HIF-1 or HIF-2 depends on availability and cooperation of HIFs with other factors recognizing their cognate elements in the promoters.

Ndrg4 enhances NGF-induced ERK activation uncoupled with Elk-1 activation.

Ndrg4 is expressed predominantly in the early postnatal rat brain and may be related to neural cell differentiation. PC12 cell lines stably expressing increased levels of Ndrg4 protein display enhanced NGF-induced phosphorylation of MEK and ERK. In contrast, the Ndrg4-C2-overexpressed PC12 cell lines showed attenuated NGF-promoted phosphorylation of Elk-1, which is a nuclear target of ERK. A reporter assay also indicated that Ndrg4-C2 suppresses Elk-1-mediated transcriptional activation and SRE reporter expression. The suppressive effect of Ndrg4-C2 on NGF-induced activation of Elk-1 was abolished by colchicine but not by cytochalasin D, suggesting that microtubules are involved in the reduced activation of Elk-1 by Ndrg4. Ndrg4 may play a role in supporting the activation of ERK and its target proteins needed for neuronal differentiation and in reducing the activation of Elk-1 implicated in cell growth.